Regulating gene expression either by increasing expression or decreasing expression is considered beneficial for treatment of human diseases. This is especially important in those diseases in which master regulatory genes have been identified. While a majority of efforts have been extended toward enhancing gene expression, down-regulating specific gene expression is equally important. A naturally occurring gene-silencing mechanism triggered by double-stranded RNA (dsRNA), designated as small interfering RNA (siRNA), has emerged as a very important tool to suppress or knock down gene expression in many systems. RNA interference is triggered by dsRNA that is cleaved by an RNase-III-like enzyme, Dicer, into 21-25 nucleotide fragments with characteristic 5′ and 3′ termini. These siRNAs act as guides for a multiprotein complex, including a PAZ/PIWI domain containing the protein Argonaute2, that cleaves the target mRNA. These gene-silencing mechanisms are highly specific and potent and can potentially induce inhibition of gene expression throughout an organism.
The last two decades have also seen tremendous progress in gene expression technology, including the continued development of both non-viral and viral vectors. The non-viral approach to gene expression involves the use of plasmid DNAs (pDNAs), which have a number of advantages, including ease of use and preparation, stability and heat resistance, and unlimited size. The plasmids do not replicate in mammalian hosts and do not integrate into host genomes, yet they can persist in host cells and express the cloned gene for a period of weeks to months.
One area that has seen renewed interest in the use of inhibitory RNA molecules is infectious diseases, and in particular mosquito-borne infectious diseases. For example, over 500 arthropod-borne viruses (arboviruses) have been identified, among which approximately 100 are harmful to humans. Such diseases, typically spread by mosquitos, impact nearly half the world's population, account for over 1 million deaths per year, and result in substantial economic losses associated with disease burden. Importantly, the US is facing increasing threats from existing and emerging mosquito-transmitted diseases, e.g. Zika virus, due to accelerated global travel, global warming, and expansion of mosquito habitat. Furthermore, for many mosquito-borne diseases, including malaria, dengue, and Zika, there are currently no vaccines. For these diseases, mosquito control remains the best option for limiting disease spread.
Traditional methods for controlling mosquito or other disease vector populations include the use of pesticides and vector control methods. Existing traditional insecticidal control methods rely upon field technicians, who may fail to find and treat many breeding sites, which can be numerous, and oftentimes inaccessible. Additional methods consist of area-wide treatment via airplane or wind-assisted dispersal from truck-mounted foggers. Unfortunately, the latter fail to treat many breeding sites and are complicated by variable environmental conditions. Additionally, surveys of natural and artificial water containers demonstrate mosquitoes and other arthropods to be highly efficient in finding, inhabiting and laying eggs in variously sized, cryptic water pools, including tree holes and gutters high above ground level. As a result, these traditional methods are inadequate to effectively control disease-carrying mosquito populations and ultimately prevent the pathogens they carry from being transmitted to human hosts.
In one specific example, the majority of vector-control strategies in the last century were based on chemical agents such as dichloro-diphenyl-trichloroethane (DDT). Although insecticides have been successfully used to control mosquitoes of the genera Aedes and Anopheles, current ecological and environmental protection standards do not allow such approaches because of the adverse effects of many insecticides on non-target species, including humans, their environmental impact, the contamination of soil and water, and the development of selective processes and subsequent mosquito resistance to insecticides. New strategies therefore had to be created to replace the use of insecticides.
In particular, the great reproductive capacity and high genomic flexibility of mosquitoes make management of these insects very difficult. Their high genomic flexibility is demonstrated by the resistance of mosquito populations to chemical and biological insecticides as well as by their ability to adapt to different environmental conditions and to climate changes. Therefore, effective alternative forms of control that can be used on a large scale and are environmentally friendly are urgently needed.
As noted above, mosquitos, acting as carriers of human pathogens, are particularly difficult to control. A typical process of vector infection begins when the pathogen enters the mosquito within a blood meal containing sufficient numbers of the pathogen to ensure some will encounter the epithelium, where the blood has been deposited in the arthropod's midgut. The pathogen must be able to cross the epithelium that has been termed the midgut infection barrier (MIB). Once in the epithelium the pathogen must replicate, cross the epithelium and escape the midgut into the hemocoel in a process termed the midgut escape barrier (MEB). The pathogen then must replicate in various mosquito tissues, but ultimately some sufficient quantity of the pathogen must invade the mosquito's salivary glands in a process overcoming the salivary gland infection barrier (SIB). There the pathogen replicates and ultimately must escape the salivary gland in the process described as the salivary gland escape barrier (SEB) upon subsequent blood feeding when it is injected into a susceptible animal host to complete the transmission cycle. This entire process can take several days to complete in the mosquito during a period called the extrinsic incubation period (EIP). Along the way, there are other arthropod related factors including various barriers to the pathogen that may also influence the pathogen and the arthropod's vector competence. The pathogen encounters arthropod digestive enzymes and digestive processes, intracellular processes, and the arthropod's immune system.
Horizontal arbovirus infection of the vector may be established upon blood-feeding of a susceptible female mosquito on a viremic vertebrate host. Within the insect vector, arboviruses have a complex life cycle that includes replication in the midgut, followed by systemic dissemination via the hemolymph and replication in the salivary glands. Transmission of an arbovirus to a naive vertebrate host during blood-feeding requires high viral titers in the saliva. Anatomical and immunological barriers affect the ability of the virus to reach such titers and thus to accomplish successful transmission to a native host. Despite efficient replication, arboviruses do not cause pathology, suggesting that the insect immune system restricts virus infection to non-pathogenic levels. Innate immunity provides the first line of defense against microbial invaders and is defined by its rapid activation following pathogen recognition by germline-encoded receptors. These receptors recognize small molecular motifs that are conserved among classes of microbes, but are absent from the host, such as bacterial cell wall components and viral double-stranded (ds) RNA. Collectively, these motifs are called pathogen-associated molecular patterns (PAMP).
For example, RNAi is one of the molecular mechanisms for regulation of gene expression generally known as RNA silencing. It has a central role in insect antiviral immunity. Notably, the RNAi response, mechanism or pathway, inhibits virus replication without causing death of the infected cell. For example, it has been shown that RNAi can eliminate Dengue virus (DENV2) from transgenic mosquitoes expressing inverted-repeat RNA to trigger the RNAi pathway against the virus. However, arboviruses are able to persistently infect vectors despite being targeted by the RNAi machinery, as shown by the presence of 21 nt virus-derived small interfering RNAs (viRNAs) in arbovirus-infected, transmission-competent mosquito vectors (Scott et al. (2010) PLOS Negl Trop Dis 4: e848; Hess et al. (2011) BMC Microbiol 11: 45).
Notably, a number of insect pathogenic viruses express a virus-encoded protein suppressor of RNAi (VSR) during replication. Expression of VSRs in insect virus-infected cells results in enhanced virus production, but in most cases these are virulence factors that greatly increase the pathogenicity of the viral infection. For example, temporally induced silencing of the RNAi machinery in Ae. aegypti led to significantly increased SINV (sindbis virus) and DENV2 (Dengue virus) titres combined with increased midgut infection and dissemination rates and a shortened extrinsic incubation period (Campbell et al. (2008) BMC Microbiol 8: 47; Sanchez-Vargas el al. (2009) PLOS Pathog 5: e1000299; Khoo et al. (2010) BMC Microbiol 10: 130). In studies involving insects, administration (e.g. by direct injections) of in vitro-synthesized dsRNA into virtually any developmental stage can produce loss-of-function mutants (Bettencourt et al. (2002) Insect Molecular Biology 11:267-271; Amdam et al. (2003) BMC Biotechnology 3: 1; Tomoyasu and Denell (2004) Development Genes and Evolution 214: 575-578; Singh et al. (2013) J Insect Sci. 13: 69).
Studies on feeding dsRNA revealed effective gene knockdown effects in many insects, including insects of the orders Hemiptera, Coleoptera, and Lepidoptera. Feeding dsRNA to E. postvittana larvae has been shown to inhibit the expression of the carboxylesterase gene EposCXEl in the larval midgut and also inhibit the expression of the pheromone-binding protein EposPBPl in adult antennae (Turner et al. (2006) Insect Molecular Biology 15: 383-391). The feeding of dsRNA also inhibited the expression of the nitrophorin 2 (NP2) gene in the salivary gland of R. prolixus, leading to a shortened coagulation time of plasma (Araujo et al. (2006) Insect Biochemistry and Molecular Biology 36: 683-693).
Similarly, direct spray of dsRNA on newly hatched Ostrinia furnalalis larvae has been reported (Wang et al. (2011) PloS One 6: e18644). The studies have shown that after spraying dsRNAs (50 ng/pL) of the DS10 and DS28 genes (i.e. chymotrypsin-like serine protease C3 (DS10) and an unknown protein (DS28), respectively) on the newly hatched larvae placed on the filter paper, the larval mortalities were around 40-50%, whereas, after dsRNAs of ten genes were sprayed on the larvae along with artificial diet, the mortalities were significantly higher to the extent of 73-100%. It was proposed through these results that in a lepidopteron insect, dsRNAs are able to penetrate the integument and could retread larval developmental, ultimately leading to death (Katoch (2013) Appl Biochem Biotechnol., 171(4): 847-73).
In mosquitoes, RNAi method using chitosan/dsRNA self-assembled nanoparticles to mediate gene silencing through larval feeding in the African malaria mosquito (Anopheles gambiae) was shown (Zhang et al. (2010) Insect Molecular Biology (2010) 19(5): 683-693). Oral-delivery of dsRNAs to larvae of the yellow fever mosquito, Ae. aegypti was also shown to be insecticidal. It was found that a relatively brief soaking in dsRNA, without the use of transfection reagents or dsRNA carriers, was sufficient to induce RNAi, and can either stunt growth or kill mosquito larvae (Singh et al. (2013), supra). Furthermore, dsRNA targeting RNAi pathway genes were described to increase Dengue virus (DENV) replication in the Ae. Aegypti mosquito and to decrease the extrinsic incubation period required for virus transmission (Sanchez-Vargas et al. (2009), supra).
A recently published RNA sequence analysis describing mosquito transcriptional profiles during DENV infection show that all transcripts representing immunity-related genes with differential accumulation in midgut samples were always more abundant in control than DENV mosquitoes, supporting the conclusion that there is a suppression of the insect immune system following infection. This result may reflect the general ‘DENV downregulation trend” observed. A similar pattern was seen in carcass samples at early time points postinfection, but the opposite was observed at 14 days post infection (dpi), reflecting a possible change in immune modulation during the course of the infection (Bonizzoni et al. (2012) PLoS ONE 7(11): e50512).
Another method for the biocontrol of vector-born pathogens includes paratransgenesis. Paratransgenesis generally refers to systems whereby symbiotic bacteria are genetically modified and reintroduced in the pathogen-bearing vector, such as mosquitos, where they express effector molecules. However, paratransgenesis has several technical limitations. For example, bacteria to be used in paratransgenesis must generally have three key components: an effector molecule that achieves the desired effect; a mechanism to display or excrete the effector molecule on the surface of the bacteria; and bacteria that can survive in the mosquito long enough to produce the expected amount of effector molecules and thereby achieve the desired effect in the mosquito. Therefore, finding such suitable bacteria that fit all of these criteria is very difficult.
Paratransgenesis is generally understood as a technique that attempts to eliminate a pathogen from vector populations through transgenesis of a symbiont of the vector. The goal of this technique is to control vector-borne diseases. The first step is to identify proteins that prevent the vector species from transmitting the pathogen. The genes coding for these proteins are then introduced into the symbiont, so that they can be expressed in the vector. The final step in the strategy is to introduce these transgenic symbionts into vector populations in the wild. Characteristics of a successful paratransgenesis system may include:                The symbiotic bacteria can be grown in vitro easily.        They can be genetically modified, such as through transformation with a plasmid containing the desired gene.        The engineered symbiont is stable and safe.        The association between vector and symbiont cannot be attenuated.        Field delivery is easily handled.A paratransgenic system is a system that can achieve paratransgenesis in a target organism.        
Identification of suitable commensal bacteria that are non-pathogenic to humans or animals among the many organisms that insects harbor, particularly in their digestive systems, is paramount for the success of a paratransgenic system. In mosquitoes, these bacteria are involved in various biological functions associated with digestion, primarily in the midgut. There is a close association between blood-dependent insects and symbiotic microorganisms that help the anabolic processes of vitellogenesis and ovogenesis. Eradication of these bacteria leads to a decline in fecundity and a slower growth rate. Interference with the digestion of proteins in mosquito blood meals can reduce fecundity and may represent a new approach for controlling mosquito populations and preventing the transmission of pathogens.
For example, the chosen bacteria should be capable of colonizing a wide variety of mosquito species so that they can be deployed in different species and isolated strains. Furthermore, the number of bacteria increases dramatically (100 to 1000 of times) after ingestion of blood, resulting in a proportional increase in the amount of effector molecules expressed and secreted by GM bacteria, leading to various possible outcomes: obstructing pathogen transmission, reducing the mosquito's vector capacity, preventing fertilization of eggs, interfering with embryogenesis and causing the death of the mosquito. These technical and physiological challenges make the development of paratransgenic systems extremely difficult. Importantly, these technical issues are such that many paratransgenic systems are neither effective nor appropriate as an effective biocontrol strategy. These difficulties may also prevent many paratransgenic systems from being appropriately scaled-up to be effective for environmental deployment. Generally, biocontrol means utilizing disease-suppressive microorganisms to eliminate, control or prevent infection, expression and/or transmission of selected pathogens.
To address the shortcomings of these traditional control methods, several other biological or non-chemical control strategies have been developed to control adult mosquito populations, including bacterial infection of mosquitoes to manipulate fitness in the wild and release of sterile males. Although these technologies have potential to control mosquito populations, they also have limitations, such as requirements for on-site rearing and limited local release of millions of engineered or infested mosquitoes at substantial costs. Furthermore, each of these strategies focuses on the control of adult mosquito populations, which must breed (requires a blood meal) for introduction and dissemination of the control strategy. Thus, there is a need for new and effective biocontrol strategies that are robust, effective over long periods of time, and cause the least possible negative environmental impact. To meet these objectives, strategies for vector control must be 1) pathogen (virus)-specific and not kill off-target organisms, 2) robust or catalytic in mode of action, 3) stable and not easily lost throughout mosquito development, 4) efficient to deliver, 5) simple to manage and low cost, and 6) self-sustainable or regenerating.
The foregoing problems regarding the biocontrol of disease-transmitting mosquito populations may represent a long-felt need for an effective and economical solution to the same. While implementing elements may have been available, actual attempts to meet this need may have been lacking to some degree. This may have been due to a failure of those having ordinary skill in the art to fully appreciate or understand the nature of the problems and challenges involved. As a result of this lack of understanding, attempts to meet these long-felt needs may have failed to effectively solve one or more of the problems or challenges here identified. These attempts may even have led away from the technical directions taken by the present inventive technology and may even result in the achievements of the present inventive technology being considered to some degree an unexpected result of the approach taken by some in the field. As will be discussed in more detail below, the current inventive technology overcomes the limitations of traditional mosquito control systems and in particular paratransgenic systems of biocontrol, while meeting the objectives of a truly effective, and scalable, vector biocontrol strategy.